Chemotaxis plays a role in the social behaviour of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium. In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.     

FrzCD, a methyl-accepting taxis protein from Myxococcus xanthus, shows modulated methylation during fruiting body formation.

The frizzy (frz) genes of Myxococcus xanthus are required to control directed motility during vegetative growth and fruiting body formation. FrzCD, a protein homologous to the methyl-accepting chemotaxis proteins from enteric bacteria, is modified by methylation in response to environmental conditions. Transfer of cells from rich medium to fruiting medium initially caused rapid demethylation of FrzCD. Subsequently, the amount of FrzCD increased, but most remained unmethylated. At about the time of mound formation (9 h), most of the FrzCD was converted to methylated forms. Dispersal of developing cells (10 h) in buffer led to the demethylation of FrzCD, whereas concentration of these cells caused methylation of FrzCD. Some mutants which were unable to form fruiting bodies still modified their FrzCD during incubation under conditions of starvation on a surface.     

A DnaK Homolog in Myxococcus xanthus Is Involved in Social Motility and Fruiting Body Formation

Myxococcus xanthus is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of M. xanthus were isolated through Tn5 transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the dnaK genes in other bacteria, this M. xanthus homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of M. xanthus.     

Spatial organization of Myxococcus xanthus during fruiting body formation

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.     

Synergism between morphogenetic mutants of Myxococcus xanthus.

Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.     

Myxococcus xanthus sasN Encodes a Regulator That Prevents Developmental Gene Expression during Growth

Myxococcus xanthus multicellular fruiting body development is initiated by nutrient limitation at high cell density. Five clustered point mutations (sasB5, -14, -15, -16, and -17) can bypass the starvation and high-cell-density requirements for expression of the 4521 developmental reporter gene. These mutants express 4521 at high levels during growth and development in an asgB background, which is defective in generation of the cell density signal, A signal. A 1.3-kb region of the sasB locus cloned from the wild-type chromosome restored the SasB⁺ phenotype to the five mutants. DNA sequence analysis of the 1.3-kb region predicted an open reading frame, designated SasN. The N terminus of SasN appears to contain a strongly hydrophobic region and a leucine zipper motif. SasN showed no significant sequence similarities to known proteins. A strain containing a newly constructed sasN-null mutation and Ω4521 Tn5lac in an otherwise wild-type background expressed 4521 at a high level during growth and development. A similar sasN-null mutant formed abnormal fruiting bodies and sporulated at about 10% the level of wild type. These data indicate that the wild-type sasN gene product is necessary for normal M. xanthus fruiting body development and functions as a critical regulator that prevents 4521 expression during growth.     

Differential effects of chemoreceptor methylation-domain mutations on swarming and development in the social bacterium Myxococcus xanthus

The soil bacterium Myxococcus xanthus is a model organism for the study of multicellular behaviour and development in bacteria. M. xanthus cells move on solid surfaces by gliding motility, periodically reversing their direction of movement. Motility is co-ordinated to allow cells to effectively feed on macromolecules or prey bacteria when nutrients are plentiful and to form developmental fruiting bodies when nutrients are limiting. The Frz signal transduction pathway regulates cellular movements by modulating cell reversal frequency. Input to the Frz pathway is controlled by the cytoplasmic receptor, FrzCD, a methyl-accepting chemotaxis protein (MCP). FrzCD lacks the transmembrane and periplasmic domains common to MCPs but contains a unique N-terminal domain, the predicted ligand-binding domain. As deletion of the N-terminal domain of FrzCD only results in minor defects in motility, we investigated the possibility that the methylation of the conserved C-terminal domain of FrzCD plays a central role in regulating the pathway. For this study, each of the potential methylation sites of FrzCD were systematically modified by site-directed mutagenesis, substituting glutamine/glutamate pairs for alanines. Four of the seven mutations produced dramatic phenotypes; two of the mutations had a stimulatory effect on the pathway, as evidenced by cells hyper-reversing, whereas another two had an inhibitory effect, causing these cells to rarely reverse. These four mutants displayed defects in vegetative swarming and developmental aggregation. These results suggests a model in which the methylation domain can both activate and inhibit the Frz pathway depending on which residues are methylated. The diversity of phenotypes suggests that specific modifications of FrzCD act to differentially regulate motility and developmental aggregation in M. xanthus.     

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.     

SigF, a new sigma factor required for a motility system of Myxococcus xanthus

A new sigma factor, SigF, was identified from the social and developmental bacterium Myxococcus xanthus. SigF is required for fruiting body formation during development as well as social motility during vegetative growth. Analysis of gene expression indicates that it is possible that the sigF gene is involved in regulation of an unidentified gene for social motility.     

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococous xanthus cells can glide both as individual cells, dependent on A dventurous motility (A motility), and as groups of cells, dependent upon S ocial motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A^- and nine new S^- mutations. In contrast with previous results, we find that subsets of A^- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S^- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S^- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S^- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S^- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S^- insertion resembles the frz^- sglA1^- mutants upon development, suggesting that this S^- gene defines a new chemotaxis component in M. xanthus.     

Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase

Myxococcus xanthus is a gliding bacterium with a complex life cycle that includes swarming, predation and fruiting body formation. Directed movements in M. xanthus are regulated by the Frz chemosensory system, which controls cell reversals. The Frz pathway requires the activity of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein, and FrzF, a methyltransferase (CheR) containing an additional domain with three tetra trico-peptide repeats (TPRs). To investigate the role of the TPRs in FrzCD methylation, we used full-length FrzF and FrzF lacking its TPRs (FrzF^CheR) to methylate FrzCD in vitro . FrzF methylated FrzCD on a single residue, E182, while FrzF^CheR methylated FrzCD on three residues, E168, E175 and E182, indicating that the TPRs regulate site-specific methylation. E168 and E182 were predicted consensus methylation sites, but E175 is methylated on an HE pair. To determine the roles of these sites in vivo , we substituted each methylatable glutamate with either an aspartate or an alanine residue and determined the impact of the point mutants on single cell reversals, swarming and fruiting body formation. Single, double and triple methylation site mutants revealed that each site played a unique role in M. xanthus behaviour and that the pattern of receptor methylation determined receptor activity. This work also shows that methylation can both activate and inactivate the receptor.     

Defects in motility and development of Myxococcus xanthus lipopolysaccharide mutants.

Five transposon Tn5 mutants of the procaryote Myxococcus xanthus had been shown previously to be defective in lipopolysaccharide biosynthesis (J. M. Fink,-M. Kalos, and J. F. Zissler, J. Bacteriol. 171:2033-2041, 1989). These mutants were studied for possible defects in gliding motility and multicellular development. Wild-type M. xanthus cells glide both as single cells and as groups of cells. We found that the Tn5 lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutant strains were slow to develop but eventually gave rise to normal, spore-filled fruiting bodies. We also had shown previously that 56 (ethyl methanesulfonate-induced and spontaneous) phage-resistant mutants were defective in lipopolysaccharide biosynthesis. We found that many of these lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutants also gave rise to normal, spore-filled fruiting bodies. We also studied several phage-resistant mutants which were lacking a side-chain carbohydrate on the lipopolysaccharide core. These mutants possessed both single-cell motility and group motility but were altered in the magnitude of gliding. These mutants were blocked early in development and could not form multicellular fruiting bodies. Several of the mutations in the developmentally aberrant strains were mapped to a single locus by using a collection of genetically linked transposons as genetic markers.     

Cell‐fate determination in Myxococcus xanthus development: Network dynamics and novel predictions

Myxococcus xanthus is a myxobacterium that exhibits aggregation and cellular differentiation during the formation of fruiting bodies. Therefore, it has become a valuable model system to study the transition to multicellularity via cell aggregation. Although there is a vast set of experimental information for the development on M. xanthus, the dynamics behind cell‐fate determination in this organism's development remain unclear. We integrate the currently available evidence in a mathematical network model that allows to test the set of molecular elements and regulatory interactions that are sufficient to account for the specification of the cell types that are observed in fruiting body formation. Besides providing a dynamic mechanism for cell‐fate determination in the transition to multicellular aggregates of M. xanthus, this model enables the postulation of specific mechanisms behind some experimental observations for which no explanations have been provided, as well as new regulatory interactions that can be experimentally tested. Finally, this model constitutes a formal basis on which the continuously emerging data for this system can be integrated and interpreted.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

Regulation of cell reversal frequency in Myxococcus xanthus requires the balanced activity of CheY‐like domains in FrzE and FrzZ

The Frz pathway of Myxococcus xanthus controls cell reversal frequency to support directional motility during swarming and fruiting body formation. Previously, we showed that phosphorylation of the response regulator FrzZ correlates with reversal frequencies, suggesting that this activity represents the output of the Frz pathway. Here, we tested the effect of different expression levels of FrzZ and its cognate kinase FrzE on M. xanthus motility. FrzZ overexpression caused a slight increase in phosphorylation and reversals. By contrast, FrzE overexpression abolished phosphorylation of FrzZ; this inhibition required the response regulator domain of FrzE. FrzZ phosphorylation was restored when both FrzE and FrzZ were overexpressed together. Our results show that the response regulator domain of FrzE is a negative regulator of FrzE kinase activity. This inhibition can be modulated by FrzZ, which acts as a positive regulator. Interestingly, fluorescence microscopy revealed that FrzZ and FrzE localize differently: FrzE colocalizes with the FrzCD receptor and the nucleoid, while FrzZ shows dispersed and polar localization. However, FrzZ binds tightly to the truncated variant FrzEΔ^CheY. This indicates that the response regulator domain of FrzE is required for the interaction between FrzE and FrzZ to be transient, providing an unexpected regulatory output to the Frz pathway.     

Spatial Simulations of Myxobacterial Development

Many bacteria exhibit multicellular behaviour, with individuals within a colony coordinating their actions for communal benefit. One example of complex multicellular phenotypes is myxobacterial fruiting body formation, where thousands of cells aggregate into large three-dimensional structures, within which sporulation occurs. Here we describe a novel theoretical model, which uses Monte Carlo dynamics to simulate and explain multicellular development. The model captures multiple behaviours observed during fruiting, including the spontaneous formation of aggregation centres and the formation and dissolution of fruiting bodies. We show that a small number of physical properties in the model is sufficient to explain the most frequently documented population-level behaviours observed during development in Myxococcus xanthus.     

Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals

The Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency. M. xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media. The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA-F putative operon and the divergently transcribed frzZ gene. FrzCD, the Frz system chemoreceptor, contains a conserved C-terminal module present in methyl-accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N-terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region. Previous work on the Frz system was limited by the unavailability of deletion strains. To understand better how the Frz system functions, we generated a series of in-frame deletions in each of the frz genes as well as regions encoding the N-terminal portion of FrzCD. Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA-CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY-CheY) are required for some but not all responses. Furthermore, deletion of approximately 25 amino acids from either end of the conserved C-terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper-reversals with no net cell movement. Surprisingly, deletion of the N-terminal region of FrzCD shows only minor defects in swarming. Thus, signal input to the Frz system must be sensed by the conserved C-terminal module of FrzCD and not the usual N-terminal region. These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP.     

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.     

Functional Organization of a Multimodular Bacterial Chemosensory Apparatus

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.